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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: J Surg Res. 2021 Aug 13;268:326–336. doi: 10.1016/j.jss.2021.06.070

Therapeutic Potential of B-1a Cells in Intestinal Ischemia-reperfusion Injury

William Royster 1,2,3, Mahendar Ochani 1, Monowar Aziz 1,2,4,†,*, Ping Wang 1,2,3,4,†,*
PMCID: PMC8678159  NIHMSID: NIHMS1727763  PMID: 34399355

Abstract

Background:

Acute mesenteric ischemia is a common surgical emergency. Restoration of blood flow is a critical objective of treating this pathology. However, many patients suffer from ischemia-reperfusion (I/R) injuries at the time of revascularization, requiring prolonged hospitalizations. B-1a cells are a subtype of B lymphocytes with roles in regulating inflammation and tissue injury by spontaneous release of natural IgM and IL-10. We hypothesized that treatment with B-1a cells protects mice from intestinal I/R.

Methods:

Mesenteric ischemia was induced in mice by placing a vascular clip on the superior mesenteric artery for 60 minutes. At the time of reperfusion, B-1a cells or PBS control were instilled into the peritoneal cavity (PerC) of mice. PerC lavage, blood, intestine, and lungs were collected 4 h after reperfusion. Serum organ injury and inflammatory markers such as ALT, AST, LDH, lactate, IL-6, as well as lung and gut histology and myeloperoxidase (MPO) were assessed.

Results:

In intestinal I/R, B-1a cell frequency and number in the PerC were significantly decreased compared to sham-operated mice. There was an increase in the serum levels of ALT, AST, LDH, lactate, and IL-6 when comparing the vehicle group with the sham group. These increases were significantly reduced in the B-1a cell treated group. B-1a cell treatment significantly decreased the intestine and lung injury scores as well as MPO content, compared to vehicle treated mice. B-1a cell treatment resulted in a reduction of apoptotic cells in these tissues. Serum IgM levels were decreased in intestinal I/R, while treatment with B-1a cells significantly increased their levels towards normal levels.

Conclusions:

B-1a cell treatment at the time of mesenteric reperfusion ameliorates end organ damage and reduces systemic inflammation through the improvement of serum IgM levels. Preserving B-1a cells pool could serve as a novel therapeutic avenue in intestinal I/R injury.

Keywords: B-1a cells, TLR4, Inflammation, Gut, Lung, Neutrophils

Introduction

Intestinal or gut ischemia is a commonly occurring surgical emergency. It often arises during abdominal and thoracic vascular surgery, small bowel transplantation, and hemorrhagic shock, leading to a high morbidity and mortality (1, 2). The primary goal in the surgical management of intestinal ischemia is the restoration of blood flow to the affected bowel, however, this frequently results in an ischemia-reperfusion (I/R) injury (3, 4). To date, a limited number of therapeutic agents have been demonstrated to provide some benefit in intestinal I/R injury (1, 5); however, none has been entirely successful.

Intestinal I/R results from an initial occlusion of blood supply to the intestines. The tissues suffer a second injury once re-perfused and oxygenation has been restored. The first injury to the tissues results from the ischemic insult resulting in tissue hypoxia subsequently leading to cellular damage and necrosis of the ischemic tissues. The second injury occurs once arterial blood flow has been restored to the tissues, causing the development of an exaggerated inflammatory response to the now damaged and necrotic tissues. (6–8). Once the vasculature has been deprived of oxygenated arterial blood, the endothelial lining loses structural integrity, resulting in increased vascular permeability, allowing for bacterial translocation into the blood stream and a subsequent systemic inflammatory response or sepsis. (9–11). The reperfusion of previously ischemic tissues results in exposure of now apoptotic and necrotic cells to a bolus of fresh immune cells and releases damage-associated molecular patters (DAMPs) such as extracellular cold-inducible RNA-binding protein (eCIRP) into circulation, further fueling the inflammatory response (3, 12–14). Immediately after reperfusion, monocytes and neutrophils migrate to the injured tissues to promote tissue repair (15). However, the excess recruitment of these immune cells leads to an overzealous inflammatory response, subsequently worsening the tissue injury (16). Intestinal I/R results in the production and release of inflammatory and oxidative molecules that damage tissue in the immediate injury as well as distant organs that were not ischemic. Acute lung injury (ALI) has been associated with intestinal I/R injury (13, 17). The pathophysiology of ALI secondary to intestinal I/R injury is mediated by the surge of proinflammatory markers released into circulation, which in turn causes recruitment of inflammatory cells, damage to endothelial cells, and smooth muscle dysfunction (14, 17). Experimentally induced ALI has been noted to closely resemble the hallmarks of acute respiratory distress syndrome (ARDS), which is a significant source of mortality in septic patients (18).

Murine B cells can be broadly categorized into two subsets, B-1 and B-2 cells (19). Follicular (FO) and marginal zone (MZ) B cells are referred to as conventional or B-2 cells (19). Murine B-1 cells have been described in the literate as expressing B220lo, CD23lo/−, CD19hi, CD43+ and CD11b+ (20, 21). Depending on the presence or absence of the marker CD5, B-1 cells can be further subdivided into either B-1a (CD5+) or B-1b (CD5−) cells (20, 21). B-1a cells are noted to reside primarily in the peritoneal cavity, but are also found in the pleural cavities, spleen, bone marrow, and blood (22). B-1a cells are the major source of natural immunoglobulin M (IgM) in unimmunologically challenged hosts. Natural IgM is notable for providing an early defense against infections by targeting less specific carbohydrate moieties commonly present on bacterial cell walls (20, 23, 24). B-1b cells have been shown to produce adaptive antibodies when exposed to T cell-independent type-2 (TI-2) antigens (25, 26). B-1a cells have been demonstrated to be a major producer of the immunoregulatory cytokine IL-10 at steady states (20, 27). During inflammation, B-1a cells of the serosal cavities (peritoneal or pleural) are relocated to the spleen or lymph nodes of the thoracic cavity and produce granulocyte-macrophage colony-stimulating factor (GM-CSF) which helps protect the host against sepsis and pneumonia (28, 29).

In addition to neutralizing invading pathogens, natural IgM from B-1a cells has also been noted to recognize and accelerate the clearance of dying cells, serving to suppress uncontrolled inflammation in states of cellular death (22, 30). B-1a cells are also notable for their production of several other immunoregulatory molecules that play crucial roles in both acute and chronic inflammation (27, 31, 32). We have recently revealed that the B-1a cell numbers were significantly decreased in sepsis, and treatment of septic mice with B-1a cells ameliorated inflammation and ALI (27, 33). Our current study dealt with the efficacy of B-1a cells to protect animals from intestinal I/R injury by controlling inflammation and tissue injury. Here, we adopted a clinically relevant model and assessed parameters for monitoring intestinal I/R-induced local and remote organ injuries after treatment with B-1a cells isolated from normal animals. Thus, treatment with B-1a cells could be a novel potential therapeutic approach in intestinal I/R injury.

Methods

Experimental animals

Male, C57BL/6 mice (8–12 weeks old, 23–27g BW) were purchased from Charles River Laboratories (Kingston, NY). Mice were housed in temperature-controlled environments and fed a standard laboratory mouse chow. Mice were given free access to food and water throughout the experiment. The mice were kept on a 12-h light/dark cycle. All animal experiments were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. This study was approved by the Institutional Animal Care and Use Committee of the Feinstein Institutes for Medical Research.

Isolation of B-1a cells

Murine B-1a cells from the peritoneal cavity (PerC) were isolated as described previously (27). Donor mice were euthanized with CO2 asphyxiation. The peritoneal cavity was instilled with 7–8 mL of PBS supplemented with 2% heat inactivated fetal bovine serum (FBS, MP Biomedicals, Irvine, CA). The abdomen was gently agitated and the lavage was aspirated. The lavage was repeated a second time. PerC lavage was centrifuged at 300 ×g for 10 minutes and resuspended in PBS with 0.5% bovine serum albumin and 2 mM EDTA buffer. B-1a cells were isolated from PerC samples in accordance with manufacturer recommendations for MACS B-1a Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Animal model of intestinal ischemia reperfusion injury

Intestinal I/R injury model in mice was performed as described previously (13, 14). Mice were anesthetized with 2% inhalational isoflurane. Hair was removed from the abdomen with clippers and the surgical field was prepped with isopropyl alcohol wipes and 10% povidone-iodine wash. An upper midline laparotomy was performed. The bowel was eviscerated and the superior mesenteric artery (SMA) was isolated. The SMA was occluded for 60 minutes with a vascular clip. At the time of clip removal, the abdomen was instilled with 1 × 106 B-1a cells freshly isolated from donor mice suspended in 500 μL of PBS or 500 μL of PBS vehicle (27, 33). The abdomen was closed in two layers and the mice were allowed to recover from anesthesia after resuscitation with a 500 μL subcutaneous bolus of normal saline. Mice were euthanized 4 hours after revascularization and peritoneal lavage, blood, lung, and small bowel tissues were collected (Supplemental Fig. 1). Sham mice underwent laparotomy with the same 1 hour of anesthesia time as the experimental animals without arterial occlusion and underwent tissue collection at the time samples were collected from experimental groups.

Assessment of B-1a cells in PerC by flow cytometry

PerC B-1a cells were determined by flow cytometry as described previously (27). PerC samples were collected from sham, vehicle, and B-1a treated mice 4 hours after mesenteric reperfusion. The PerC was instilled with 7–8 mL of PBS supplemented with 2% heat-inactivated FBS. The abdomen was gently agitated and the PerC lavage was aspirated. This process was repeated for a second time. PerC lavage samples were centrifuged at 300 ×g for 10 minutes and re-suspended in 500 μL of FACS buffer. A total of 1 × 10 6 PerC cells were suspended in 500 μL of FACS buffer and stained with PE-Cy7 anti-mouse CD23 antibody (clone: B2B4, BioLegend, San Diego, CA), PE anti-mouse B220 antibody (clone: RA3-6B2, BD Biosciences, San Jose, CA), and PerCP-Cy5.5 anti-mouse CD5 antibody (clone: 53-7.3 BioLegend, San Diego, CA) for 60 min at 4 °C. DAPI (Thermo-Fisher, Waltham, MA) was added 15 minutes prior to assessment of cells with flow cytometry. Unstained cells were used to establish control voltage settings and single color compensation was established with UltraComp eBeads (Thermo-Fisher, Waltham, MA). Acquisition was performed on 100,000 events using a BD FACSymphony flow cytometer (BD Biosciences, San Jose, CA) and data were analyzed with FlowJo software (Tree Star, Ashland, OR).

Determination of organ injury markers

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and lactate were determined using specific colorimetric enzymatic assays (Pointe Scientific, Canton, MI) according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay (ELISA)

Serum IL-6 was analyzed according to manufacturer’s (Biosciences, San Jose, CA) instructions. In brief, a total of 25 μL of serum isolated from sham, vehicle, and treatment mice diluted in assay diluent to a final volume of 100 μL were loaded into IL-6 ELISA wells and incubated for 60 minutes at room temperature. Wells were washed and 100 μL of HRP-labeled conjugate was added to each well and incubated for 60 minutes at room temperature. The wells were washed and required volume of detection reagent was added to each well and allowed to develop color for 30 minutes. Finally, OD was measured at 450 nm wavelength and the concentrations of samples were calculated from the standard curve. Serum IgM levels were analyzed according to the manufacturer’s (Bethyl Laboratories, Inc., Montgomery, TX) instructions.

Myeloperoxidase (MPO) assay

A total of 50–100 mg of liquid nitrogen-based powdered lung and intestinal tissues were homogenized in KPO4 buffer containing 0.5% hexa-decyl-trimethyl-ammonium bromide (Sigma-Aldrich, St. Louis, MO) using a sonicator with the samples placed on ice. After centrifuging, the supernatant was diluted in reaction solution which contains O-Dianisidine dihydrochloride (Sigma-Aldrich) and H2O2 (Thermo-Fisher Scientific, Waltham, MA) as a substrate. Rate of change in optical density (ΔOD) between 1 and 4 min was measured at 460 nm to calculate MPO activity.

Lung and intestinal tissue histology

Formalin fixed and paraffin embedded lung and intestinal blocks were sectioned at 5 μm thickness and placed on glass slides. Lung and intestinal tissue samples were stained with hematoxylin and eosin (H&E). Slides were analyzed using bright field microscopy in a blinded manner. Lung injury was assessed in accordance with a scoring system established by the American Thoracic Society (34). Scores ranged from 0 to1 and were based on the presence of neutrophils in the alveolar and interstitial spaces, hyaline membranes, proteinaceous debris in the airspaces, and alveolar septal thickening. Scores were calculated at 200× magnification. Intestinal organ injury was scored based on a system created specifically for murine intestinal ischemia reperfusion models (35). Scores ranged from 0 to 4 and were based on the presence of multiple factors, including epithelial cell degeneration, decrease of villus to crypt ratio, effacement of the villi, and transmural necrosis.

Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay

The presence of apoptotic cells in lung and intestinal tissue sections were determined using a TUNEL assay kit (Roche Diagnostics, Indianapolis, IN). Tissue sections were fixed in 10% phosphate buffered formalin and were then embedded into paraffin and sectioned at 5 μm following standard histology procedures. Sections were dewaxed in xylene, rehydrated in serial dilutions of ethanol, and equilibrated in Tris buffered saline (TBS). The sections were then digested with 20 μg/mL proteinase K for 20 min at room temperature. The tissue sections were then washed and incubated with a cocktail containing terminal deoxynucleotidyl transferase enzyme and fluorescence labeled nucleotides in accordance with manufacturer’s instructions. Slides were examined under a fluorescent microscope (Nikon Eclipse Ti-S, Melville, NY).

Statistical analysis

All statistical analyses were performed and the figures were prepared with GraphPad Prism version 9.0 software (GraphPad Software, La Jolla, CA). Data are expressed as mean ± standard deviation (SD). Normality of continuous data were assessed with the Shapiro-Wilk test. Comparisons between two groups were performed with a two-tailed Student’s t-test (parametric). Comparisons between multiple groups were analyzed using a one-way analysis of variance (ANOVA), followed by Student-Newman-Keuls (SNK) comparison test. The statistical significance was set at p < 0.05.

Results

B-1a cells are decreased after intestinal I/R injury

PerC cells from sham and intestinal I/R mice were gated based on FSC-A vs. SSC-A. Singlets were then gated based on FSC-A vs. FSC-H. Live cells were then gated out as DAPI negative. B-1a cells were identified based on the phenotype of CD23−B220loCD5int (Fig. 1A–B). PerC B-1a cell frequency was significantly decreased by relative 76% (11.14 ± 1.36% vs. 2.64 ± 0.9%) (Fig. 1C) and absolute number was significantly decreased by 90% (12.1 × 104 ± 2147 cells vs. 1.2 × 104 ± 1036 cells) 4 h after intestinal I/R compared to sham operation (Fig. 1D).

Figure 1: Intestinal I/R causes a reduction of the frequency and number of PerC B-1a cells.

Figure 1:

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After 4 h of intestinal I/R, cells from the peritoneal cavity of sham and intestinal I/R mice were collected. Peritoneal cells were stained with DAPI, CD23, B220 and CD5 Abs and subjected to flow cytometric detection of B-1a cells frequencies. Representative dot blots of the gating strategy of B-1a cells of (A) sham and (B) intestinal I/R mice are shown. Status of B-1a cell’s (C) frequencies and (D) numbers in intestinal I/R are shown. Data are expressed as means ± SD (n=5–6 mice/group). The groups were compared by Student’s t-test (*p<0.05 vs. sham mice).

B-1a cell treatment ameliorates serum organ injury and inflammatory markers after mesenteric reperfusion

MACS sorted B-1a cells were adoptively transferred into ischemic mice to replenish the PerC B-1a pool at the time of mesenteric reperfusion. Serum markers of systemic injury and inflammation such as ALT, AST, LDH, lactate, and IL-6 were all significantly decreased in B-1a-treated mice compared to PBS-treated mice 4 h after mesenteric reperfusion by mean values of 44%, 55%, 56%, 42%, and 54%, respectively (Fig. 2A–E).

Figure 2: Treatment with B-1a cells attenuates serum levels of organ injury markers and IL-6 after intestinal I/R.

Figure 2:

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Mice were randomly assigned to sham laparotomy, treatment, or vehicle group. Intestinal I/R was induced in mice via SMA occlusion for sixty minutes. Treatment mice received an intraperitoneal instillation of 1 × 106 B-1a cells at the time of reperfusion. Vehicle groups received an equivalent volume of PBS. Four hours after recovery from anesthesia, mice were sacrificed, and serum collected for analysis. (A) ALT, (B) AST, (C) LDH, (D) lactate, and (E) IL-6 were determined using specific colorimetric enzymatic and ELISA assays. Data are expressed as means ± SD (n=4–6 mice/group). The groups were compared by one-way ANOVA and Student-Newman-Keuls (SNK) method (*p<0.05 vs. sham and #p<0.05 vs. vehicle mice).

B-1a cell treatment results in decreased intestinal injury and apoptosis after mesenteric reperfusion

Gut tissues of sham, vehicle or B-1a cell-treated I/R mice were collected and preserved in 10% formalin. The tissues were paraffin embedded, sectioned 5-μm thick, and stained with H&E. Analysis of the tissues under light microscopy revealed a significant reduction in organ injury score (2.67 ± 0.52 vs. 1.5 ± 0.54) (Fig. 3A–B). Intestinal sections from B-1a treated mice had less effacement of the villi and no evidence of transmural necrosis. Analysis of MPO contents of the gut tissues showed a significant decrease after B-1a treatment compared to vehicle treatment (28.9 ± 3.15 U/g vs. 18.9 ± 5.22 U/g) (Fig. 3C). The 34% decrease in MPO for B-1a treated intestinal tissues suggests a blunting of neutrophil recruitment into the tissues. Total apoptotic cells were noted to be significantly decreased in B-1a treated intestinal tissues based on TUNEL assay (58 ± 10.8 cells/field vs. 26 ± 8 cells/field) (Fig. 4A–B).

Figure 3: B-1a cells protect mice from intestinal injury caused by intestinal I/R.

Figure 3:

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Four hours after reperfusion, gut tissues were collected for histological analysis. (A) Representative images of H&E stained gut tissue at 200×. Scale bar: 100 μm. (B) Gut injury scores ranged from zero to four were based on the assessment of villus-to-crypt ratio, lymphocytic infiltrates, epithelial degeneration/necrosis, erosions, glandular dilatation, and transmural changes. Data are expressed as means ± SD (n=4–6 mice/group) and compared by one-way ANOVA and Student-Newman-Keuls (SNK) method (*p<0.05 vs. sham and #p<0.05 vs. vehicle mice). (C) Myeloperoxidase (MPO) activities in gut tissues were assessed spectrophotometrically. Data are expressed as means ± SD (n=2–4 mice/group) and compared by one-way ANOVA and Student-Newman-Keuls (SNK) method (*p<0.05 vs. sham and #p<0.05 vs. vehicle mice).

Figure 4: Apoptosis in the intestine after intestinal I/R is decreased in B-1a cell-treated mice.

Figure 4:

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Intestine tissue from sham, vehicle, and B-1a cell treated intestinal I/R mice were harvested 4 hours after 60-minute ischemia to evaluate for apoptosis. (A) Representative images of terminal deoxynucleotidyl TUNEL (green fluorescence) and nuclear counterstaining (blue fluorescence). Magnification 100×. (B) Number of TUNEL positive cells per 100× magnified field. Data are expressed as means ± SD (n=4–6 mice/group). Groups were compared by one-way ANOVA and Student-Newman-Keuls (SNK) method (*p<0.05 vs. sham and #p<0.05 vs. vehicle mice).

B-1a cell treatment protects mice from lung injury and reduces apoptosis after mesenteric reperfusion

Histological images of lung tissue indicated severe lung injury based on the recruitment of neutrophils into the alveolar and interstitial spaces and the presence of hyaline membranes and proteinaceous debris in the airspaces after mesenteric reperfusion injury. B-1a cell treatment was able to significantly reduce the organ injury score by 52% (Fig. 5A–B). There was a significant decrease in MPO content in the B-1a treated lung tissues compared to vehicle treated I/R mice (38.2 ± 4.2 U/g vs. 19.9 ± 8.6 U/g) (Fig. 5C). Mesenteric ischemia reperfusion resulted in a significant number of apoptotic cells in the lungs. Treatment with B-1a cells showed a 58% decrease in apoptotic cells compared to vehicle treated mice (21 ± 4.63 cells/field vs. 9 ± 3.14 cells/field) (Fig. 6A–B).

Figure 5: B-1a cells protect mice from acute lung injury caused by intestinal I/R.

Figure 5:

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Four hours after reperfusion, lung tissues were collected for histological analysis. (A) Representative images of H&E stained lung tissue at 200×. Scale bar: 100 μm. (B) Lung injury scores ranged from zero to one were based on the presence of proteinaceous debris in the airspaces, the degree of septal thickening, and neutrophil infiltration in the alveolar and interstitial spaces. Data are expressed as means ± SD (n=4–6 mice/group) and compared by one-way ANOVA and Student-Newman-Keuls (SNK) method (*p<0.05 vs. sham and #p<0.05 vs. vehicle mice). (C) Myeloperoxidase (MPO) activities in lungs were assessed spectrophotometrically. Data are expressed as means ± SD (n=2–4 mice/group) and compared by one-way ANOVA and Student-Newman-Keuls (SNK) method (*p<0.05 vs. sham and #p<0.05 vs. vehicle mice).

Figure 6: Apoptosis in the lungs after intestinal I/R is decreased in B-1a cell-treated mice.

Figure 6:

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Lung tissue from sham, vehicle and B-1a cell treated intestinal I/R mice were harvested 4 hours after 60-minute ischemia to evaluate for apoptosis. (A) Representative images of terminal deoxynucleotidyl TUNEL (green fluorescence) and nuclear counterstaining (blue fluorescence). Magnification 200×. (B) Number of TUNEL positive cells per 200× magnified field. Data are expressed as means ± SD (n=4–6 mice/group). Groups were compared by one-way ANOVA and Student Newman Keuls (SNK) method (*p<0.05 vs. sham and #p<0.05 vs. vehicle mice).

The adoptive transfer of B-1a cells at the time of mesenteric reperfusion resulted in a preservation of serum IgM levels

Natural IgM produced by the B-1a cells play several physiological roles. Among them, are to facilitate the clearance of apoptotic cells (20, 30). Four hours after mesenteric reperfusion, serum IgM levels were significantly decreased in vehicle treated mice. The adoptive transfer of B-1a cells resulted in a 33% improvement in serum IgM levels (116.6 ± 19.4 μg/mL vs. 173.4 ± 30.2 μg/mL) (Fig. 7).

Figure 7: Treatment with B-1a cells restores the serum levels of IgM after intestinal I/R.

Figure 7:

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Mice were randomly assigned to sham laparotomy, treatment, or vehicle group. Intestinal I/R was introduced in mice via SMA occlusion for 60 minutes. Treatment mice received an intraperitoneal instillation of 1 × 106 B-1a cells at the time of reperfusion. Vehicle groups received an equivalent volume of PBS. Four hours after recovery from anesthesia, mice were sacrificed, and serum collected for the analysis of IgM by ELISA. Data are expressed as means ± SD (n=4–6 mice/group). The groups were compared by one-way ANOVA and Student-Newman-Keuls (SNK) method (*p<0.05 vs. sham and #p<0.05 vs. vehicle mice).

Discussion

B-1a cells exhibit an immunoregulatory function, which is mediated through the production of the anti-inflammatory cytokine IL-10, natural IgM, and GM-CSF (20, 31). We previously demonstrated the beneficial role of B-1a cells in sepsis as mediated through IL-10 (27, 33). Others also reported the beneficial role of B-1a cells in sepsis mediated through natural IgM and GM-CSF (28, 29). However, the role of B-1a cells in intestinal I/R injury has not been studied before. In the present study, we revealed that during experimental intestinal I/R, the frequency and number of B-1a cells in the peritoneal cavity, where most of the murine B-1a cells reside, were significantly decreased. Adoptive transfer of B-1a cells of syngeneic healthy mice significantly reduced organ injury markers, ALT, AST, LDH, and lactate and the pro-inflammatory cytokine IL-6 in the serum. In particular, the treatment with B-1a cells attenuated intestinal injury score and reduced MPO production, reflecting less neutrophil accumulation in the injured gut and reduced apoptosis. In addition to the local tissue injury, treatment with B-1a cells significantly reduced remote organ (lung) injury. MPO levels and apoptotic cells were also decreased in the lungs of intestinal I/R mice treated with B-1a cells. We had previously demonstrated that the use of B-2 cells as a control treatment at the time of sepsis induction did not provide any benefit to survival or plasma inflammation or injury markers (27). These findings demonstrate that B-1a cells may serve as a novel therapeutic avenue in intestinal I/R injury.

Based on our previous research with B-1a cells in murine sepsis, we set the concentration of B-1a cells to treat gut I/R mice. In our earlier study, we treated septic mice with 0.5 million B-1a cells obtained from normal healthy syngeneic mice via intraperitoneal injection at the time of sepsis induction by CLP operation (27, 33). We found that adoptive transfer of 0.5 million B-1a cells significantly decreased the lung damage and the levels of pro-inflammatory cytokines in the blood. With this effective dose in mind, we first treated gut I/R mice with 0.5 million B-1a cells in the present study. We noticed no reduction of pro-inflammatory cytokines and injury markers in the blood. As a result of the refractory findings with the dose of 0.5 million of B-1a cells in our gut I/R model, we increased the number of B-1a cells transferred to gut I/R mice from 0.5 to 1 million B-1a cells and found favorable outcomes. We used only male mice in this study. A gender-specific difference in morbidity and mortality and other outcomes in females has been reported in critically ill patients with sepsis (36). It has been reported that male and female sex hormones exhibit diverse immune-modulating functions in various diseases (37). Given the impact of sex on innate immune function, only male mice were used to generate reliable and consistent findings. However, sepsis is a disease of all ages and genders, where females and males are equally vulnerable to develop sepsis in response to infection. Therefore, given the equal importance of studying female mice, we admitted it as a limitation of our study that might impact the interpretation of the finding.

The decrease of the frequencies and numbers of B-1a cells in the peritoneal cavity following intestinal I/R injury might render the affected mice susceptible to inflammation as these cells play a pivotal role in regulating excessive inflammation. B-1a cells are resistant to apoptosis (38, 39), thus the decrease of B-1a cells after intestinal I/R might instead involve migration and differentiation (27, 40, 41). Using green fluorescent protein (GFP) tagged B-1a cells to track their migration, we recently showed that following induction of sepsis, B-1a cells in the peritoneal cavity migrate to the spleen and lymph nodes (27). A recent study showed that injection of bacteria or LPS into the peritoneal cavity promoted PerC B-1a cells to migrate toward the spleen where they were transformed into plasma cells to produce more immunoglobulin (41). Migration of PerC B-1 cells has been shown to be associated with the upregulation of CXCR4 and increased migratory response to CXCL12 (40). Another study revealed that the B-cell-specific loss of CXCR4 decreases B-1a number and IgM production within the bone marrow (BM), a niche of natural IgM, resulting in decreased plasma IgM (42). Conversely, B-1a cell-specific overexpression of CXCR4 in vivo is associated with increased B-1a localization to the BM. As such, it is possible that depending on the expression of CXCR4 on B-1a cells, they may migrate to the BM.

Natural IgM secreted from B-1a cells serves multiple different functions (31, 43, 44). Murine B-1a cell natural IgM is not high affinity like the IgG secreted by plasma cells. B-1a cell-derived natural IgM are also noted to be polyreactive and are able to recognize phosphorylcholine (PC), common component of the cell wall of gram-positive bacteria. Natural IgM also tend to target components of the membranes of bacteria, apoptotic cells, and oxidized low-density lipoprotein (OxLDL) (22, 44). Natural IgM are capable of directly neutralizing pathogens, activating the complement cascade, or opsonizing the antigen resulting in phagocytosis by innate immune cells and/or Ab-dependent cell-mediated cytotoxicity (45). Natural Abs can also aid in the clearance of dead and dying (30, 31, 43). The clearance of the cells ameliorates the release of inflammatory markers that could set off an inflammatory cascade in response to dead and dying tissues. In the present study, we found less apoptotic cell infiltration in the injured gut mucosa as well as in the lungs in B-1a cell treated intestinal I/R mice. This indicates that B-1a cell treatment either reduces apoptosis or increases the clearance apoptotic cells. Given the anti-inflammatory effects of B-1a cells, the overall inflammation and tissue injury were significantly reduced, which might result in decreased apoptosis or necrosis. The increased levels of IgM in B-1a cell treated mice might remove apoptotic cells by B-1a cell-derived IgM. However, a recent report suggested that deposition of IgM within reperfused tissues correlated with pathogenesis after treatment of mice reconstituted with IgM isolated from a single pathogenic IgM clone (46). The sequence analysis of pathogenic IgM identified an apparent usage of germ-line VH and VK genes in its light chains, respectively. Therefore, given this controversy of differential findings on IgM on intestinal I/R, further studies are needed to confirm the impact of B-1a cell IgM on intestinal I/R.

Several molecules have been shown to influence B-1a cell function. eCIRP, a novel DAMP, has recently been shown to be upregulated in models of sepsis, hemorrhagic shock, and ischemia reperfusion injuries (47). eCIRP is capable of binding multiple different receptors such as TLR4, TREM-1, and IL-6R. Upon binding to these receptors, eCIRP is able to induce profound inflammatory responses from multiple different cell types (48, 49). eCIRP may play a critical role in how B-1a cells respond to states of sepsis or I/R injuries. Upon activation of murine peritoneal B-1a cells with LPS, peritoneal B-1a cells migrate to the spleen and produce GM-CSF, which in turn induces B-1a cells in an autocrine manner to produce more IgM to combat pathogens (28, 29). Sialic acid binding Ig-like lectin G (Siglec-G) plays a critical role in BCR-mediated B cell activation and development (50). Siglec-G-deficient mice produce increased numbers of B-1a cells and serum IgM levels (51). In a murine model of atherosclerosis, Siglec-G knockout mice were shown to be protective though the increased release of IgM which neutralized oxLDL (51). However, it is currently unknown if the IgM released from Siglec-G knockout B-1a cells will have the same repertoire skew, affinity, and or polyreactivity as natural IgM from wild-type B-1a cells. It has been previously shown that in aged animals, B-1a cell number and IgM levels are declined, rendering the aged animals to become susceptible to bacterial infection (52). Future studies of intestinal I/R in aged animals after treatment with B-1a cells will provide deeper insights on the impact of these cells in various age group of subjects suffering from gut I/R. The role of other regulatory B cells like the B10 cells has been reported in chronic intestinal diseases such as inflammatory bowel disease (20, 53). Future studies with B10 cells on intestinal I/R would be of great potential.

In conclusion, our study has demonstrated that restoration of the B-1a cell pool in the PerC is able to reduce systemic inflammatory markers, ameliorate evidence of end organ damage, and augment serum IgM levels which could help clear apoptotic cells as well as pathogenic bacteria from gut translocation after mesenteric I/R injury. The phenotype of human B-1a cells has been identified (54). Given that murine B-1a cells have been shown to be beneficial in mesenteric I/R injury, further research into the role of human B-1a cells in treating acute mesenteric ischemia is warranted.

Supplementary Material

1

Acknowledgements

We thank Zhimin Wang and Yongchan Lee of the Center for Immunology and Inflammation, Feinstein Institutes for Medical Research for their technical support.

Grants

This study was supported by the National Institutes of Health (NIH) grants R01HL076179 (PW), R35GM118337 (PW) and R01GM129633 (MA).

Footnotes

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Competing interests

All authors declared that they have no competing interests.

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